The dry mill ethanol production process primarily utilizes whole ground grain, e.g., corn or milo, or fractionated corn. In a typical dry milling process, the entire corn kernel or other starchy grain is first milled to a specific particle size (<1.0 mm) and then processed without further separating the various components of the grain. The milled grain is made into a slurry (25-40% dissolved solid (DS) grain) with hot cook water (>90° C.), and mixed well in a mix box. An initial dose of thermostable liquefying alpha amylase is added to the slurry in the mix box before the slurry is transferred to a primary liquefaction tank.
Then the pH of the partially gelatinized starch slurry is conventionally adjusted to greater than pH 5.8 using ammonia, and then incubated with a thermostable alpha amylase at 85-86° C. for 15-20 minutes before being sent through a jet cooker maintained at 105-108° C. for a holding time of 3-5 minutes. Following jet cooking, the gelatinized starch slurry is held in a cook tube at high pressure for 8 to 10 minutes to complete gelatinization, and then flashed to atmospheric pressure and maintained the temperature at about 85° C.
A second dose of thermostable alpha amylase is typically added to complete the liquefaction of starch as the slurry is held at the elevated temperature for 90 to 120 minutes and then sent to a series of heat exchangers for reducing the temperature to 32° C. prior to fermentation. The high temperature also reduces the high risk of microbial contamination of the mash. Following liquefaction, the pH of the mash is decreased to less than pH 5.2 using dilute sulfuric acid and then cooled to 32° C. prior to fermentation. This process is diagramed in FIG. 1, which indicates the steps where pH adjustments are necessary.
In a dry mill ethanol process, the whole ground grain is generally mixed with fresh water, condensate water, and thin stillage (generally called cook water or backset) at 10-50% to produce a mash with a DS content ranging from 25% to 45%. The natural pH of the whole ground grains, such as corn or milo/sorghum, in water ranges from 5.5 to 6.2 depending upon the length of storage of the grain and extent of microbial infection. However, the pH is lowered to pH 4.8 to pH 5.2 when the ground grains are mixed with varying amount of thin stillage. The pH of the thin stillage also varies significantly depending on the particular processing plant, with typical pH value ranging from 3.8 to 4.5. Some ethanol producers add acids, e.g., to lower the pH in the beer and to reduce the risk of microbial contamination prior to distillation, thereby lower the pH.
To illustrate this point, the effect on final pH of adding of thin stillage from a commercial ethanol plant at different ratios in the make-up water added to a whole ground corn slurry was recently studied at an ethanol production plant in Monroe, Wis., USA. As shown in Table 1, the more thin stillage that is used as make-up water, the lower the final pH of the slurry.
TABLE 1Effect of thin stillage concentrations on the final pH of a whole groundcorn slurry (32% DS corn), stirred for 2 hours at 32° C. (155° F.).Final pH of 32% DS wholeThin stillage, (% w/w)ground corn slurry05.52205.29405.16505.09605.05804.981004.94
It is generally important for ethanol production plants to use cook water as make-up water in the slurry tank to conserve water; therefore this practice should not necessarily be discouraged. However, current commercially-available thermostable alpha amylase enzymes that are used to convert granular starch in whole ground grains into soluble dextrins during primary liquefaction are not stable below pH 5.6 at the elevated temperatures used in the process. Providing a suitable environment for the alpha amylases, therefore, necessitates the adjustment of the pH to pH 5.8 to pH 6.0 using suitable alkali reagents, such as sodium hydroxide, sodium carbonate, or ammonia. This pH adjustment is more than just an added step because it typically adds a significant amount of ions, e.g., sodium to the fermentation medium, which may impact the growth of microorganisms during subsequent processing steps, e.g., the growth of yeast during fermentation.
Starting the yeast fermentation at a higher pH due to the addition of alkaline reagents increases the risk of microbial contamination. As a result, alcohol producers generally reduce the pH after liquefaction to less than pH 5.0 (e.g., pH 4.2 to 4.5) using dilute sulphuric acid. In addition to adding yet another pH adjustment step, the addition of sulphuric acid results in a slurry with a higher sulphur content, which can result waste disposal problems and raise environmental concerns. Another problem associated with using sulphuric acid for pH adjustment results in DDGS, an animal feed component with high sulphur content.
It is apparent that the need to adjust the pH of a slurry or mash to accommodate commercially-available enzyme preparations increases the numbers of steps required for grain processing and introduces ions and other chemicals into the slurry or mash that can adversely affect microorganism growth, the quality of the co-product, and the ease of disposing of process waste materials.
Another problem with conventional grain processing methods involves phytic acid (i.e., phytate, myo-inositol hexakis-phosphate, or IP6). Phytate is the primary storage form of phosphate in cereals/grains and oil seeds (see, e.g., Graf, E. (ed.) “Phytic acid Chemistry and Applications” (1986) Pilatus Press, Minneapolis, USA). Phytate consists of myo-inositol ring and six symmetrically distributed phosphate groups. Phytate is generally considered an undesirable component of grain and cereals for use in feed formulations because the phosphate is unavailable to monogastric animals due to its limited digestibility. Phytate is also known to bind essential minerals, such as zinc, iron, calcium, magnesium and proteins resulting in a reduction in their bioavailability (Maenz D. et al. (1997) Anim. Feed. Sci. 72:664-68; Ritter, M. et al. (1987) J. Food Sci. 52, 325-41). Phytate and other myo-inositol phosphate esters have also been shown to exhibit alpha-amylase inhibitory effects with respect to the hydrolysis of starch (Knuckles B. and Betschart, A. (1987) J. Food Sci. 52, 719-21).
Phytate hydrolyzing enzyme (i.e., phytase; myo-inositol hexaphosphate phosphohydrolase, E.C. 3.1.3.8) hydrolyses phytic acid into inorganic phosphates and inositol mono-to-penta-phosphates. The enzyme is widely distributed in plants, micro-organisms and animal tissues (Wodzinski, R. and Ullah, A. (1996) Advances in Applied Microbiology 42:264-303; Dvorakova J. (1998) Folia Microbiol 43:323-338). Plant phytases generally exhibit activity between pH 4.5 to 6.5, with a temperature optimum of 55° C. Thus, the processing conditions of animal feed formulation generally results in the complete inactivation of the endogenous phytases. As a consequence, microbial phytases are often used in feed formulations. Commercially available microbial phytases include Phyzyme™ XP 5000 from Genencor, Finase™ from AB Enzymes, GODO PHY™ from Godo Shusei Japan, Allzyme™ Phytase from Altech, Natuphos™ from BASF, Ronozyme™ P from DSM/Novozyme.
Pretreatment of cereals and grains with phytases to reduce phytic acid content has also been reported. For example, U.S. Pat. No. 4,914,029 describes a process for treating corn or sorghum kernels with phytase under steeping conditions in the presence of sulphur dioxide to eliminate or greatly reducing the phytin content in corn steep liquor. An enzymatic process using phytase for producing phytate free or low phytate soy protein isolate/concentrate is described in European Patent Pub. No. EP 380 343. U.S. Pat. No. 5,756,714 further describes enhanced hydrolysis of starch by an alpha-amylase under liquefaction conditions by pretreating the starch slurry with phytase. International Pat. Pub. No. WO 98/11788 describes a method for reducing the phytin content of the cereals product by subjecting to a combined wet steeping and dry steeping in at least two successive cycles whereupon activating the endogenous phytase for hydrolyzing the phytic acid. Finally, U.S. Pat. Pub. No. 2005/0272137 describes an improved fermentation process wherein phytic acid containing material is fermented in the presence of phytase.
The presence of phytic acid in grains impacts the ethanol production process by increasing the cost of waste disposal, reducing the amount of thin stillage that can be recycled, binding trace metals necessary for the growth of microorganisms, decreasing the activity of proteolytic enzymes, and reducing the rate and efficiency of starch hydrolysis by inhibiting alpha amylases.
It is therefore apparent that the need exists to reduce the amount of phytate present in grains and cereal-derived product.